Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus
2019
Аутори:
Pajić, TanjaStevanović, Katarina
Todorović, Nataša
Krmpot, Aleksandar
Rabasović, Mihailo
Jelenković, Branislav
Živić, Miroslav
Остала ауторства
Grbović Novaković, JasminaNestorović, Nataša
Rajnović, Dragan
Тип документа:
Конференцијски прилог (Објављена верзија)
,
© 2019 Institute for Biological Research "Siniša Stanković" – National Institute of Republic of Serbia, University of Belgrade
Метаподаци
Приказ свих података о документуАпстракт:
Label-free two photon nonlinear microscopy is well established as a powerful tool for monitoring
metabolic state of the various cell types due to its non-perturbative nature and fairly low phototoxicity,
while application of third harmonic generation (THG) for three-dimensional (3D) cell and tissue
microscopy was enabled more recently. THG occurs at structural interfaces, such as local transitions
of the refractive index, most generally speaking at interfaces that are formed between aqueous
interstitial fluids and lipid-rich structures. Here we present preliminary data obtained by capturing
both THG and optical redox ratio signal from the same regions of the hyphae of Phycomyces
blakesleeanus.
Label- free metabolic intravital microscopy through application of both THG and NAD(P)H+/FAD+
autofluorescence ratio was used in alternating sequence on the same field of view on a fungal cells of
a model filamentous fungus Phycomyces blakesleeanus. The glass coverslip with collagen coating
bearing unstained hyphae was mounted on custom built microscope. Laser beams for both imaging
modalities were focused with the same objective lens, Zeiss Plan Neofluar 40x1.3. The
autofluorescence of NAD(P)H was excited by Ti:Sa laser pulses at 730 nm, 160 fs duration and signal
was collected through 479/40 filter, while for autofluorescence of FAD we used excitation by the
same laser pulses at 860 nm, 160 fs duration and 530/43 filter.
For THG, we used 1040 nm, 200 fs pulses from Yb KGW laser, and detection was performed by
PMT through Hoya glass UV filter with peak transmission at 340nm. As a control for perturbation of
optical redox ratio, rothenone (complex I inhibitor) was applied in some experiments. Nile red
staining was used to confirm that the brightest structures of round shape in THG images consist of
lipids and probably represent lipid droplets that serve as energy deposits in hyphea.
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200007 (Универзитет у Београду, Институт за биолошка истраживања 'Синиша Станковић') (RS-MESTD-inst-2020-200007)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200178 (Универзитет у Београду, Биолошки факултет) (RS-MESTD-inst-2020-200178)
У:
- Grbović Novaković J, Nestorović N, Rajnović D, editors. Proceedings: 14th Multinational Congress on Microscopy; 2019 Sep 15-20; Belgrade, Serbia. Belgrade: Institute for Biological Research "Siniša Stanković"– National Institute of Republic of Serbia, University of Belgrade; 2019. p. 122.